Double stranded RNA constructs for aphid control

ABSTRACT

Disclosed are specific aphid dsRNA constructs that target either Chloride Intracellular Channel (CLIC) gene expression or Sucrase gene expression. Also disclosed is the use of dsRNA constructs of a CLIC gene to interfere with critical functions of CLIC gene peptide products. A novel method to develop nucleic acid control for pest management is also disclosed. Also disclosed is the use of dsRNA constructs to interfere with critical functions of Sucrase gene peptide products.

CROSS-REFERENCE TO RELATED APPLICATION

This present application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Ser. No.: 62/040,714, which was filed on Aug. 22, 2014, andis hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to double stranded RNA constructs to inhibit theexpression of either Chloride Intracellular Channel protein (CLIC) orSucrase to induce mortality in aphid species, including but not limitedto Diuraphis noxia, Myzus persicae, and Schizaphis graminum.

BACKGROUND OF INVENTION

There are currently 5000 species of Aphididae with over 100 specieseconomically important as pests of crops (Blackman & Eastop 2006, 2007).Aphids are phloem sap feeders that damage the plants through the removalof carbohydrates and amino acids and by injecting phytotoxic saliva andvectoring plant diseases while feeding. The Russian wheat aphid,Diuraphis noxia, and the greenbug, Schizaphis graminum, are globallydistributed and significant pests of cereals, causing losses exceeding$250 million/year in the mid-western United States alone (Webster 1994,Morrison and Peairs 1998). M. persicae attacks over 200 species ofplants and is known by many names including green peach aphid orpeach-potato aphid. Plant damage occurs mainly from the aphid'stransmission of viruses lethal to many vegetables and tobacco (Eastop1977. Blackman and Eastop 2000). The low profit margins in cropproduction and frequent insecticide use required to mitigate lossescaused by aphids are economically unsustainable.

Chemical pesticides such as pyrethrins and pyrethroids are the mostcommon means of controlling aphids. However the use of traditionalchemical pesticides has disadvantages, including non-target effects onneutral or beneficial insects, as well as other animals. Chemicalpesticide usage also can lead to chemical residue run-off into streamsand seepage into water supplies resulting in ecosystem/environmentdamage. In addition, animals higher in the food chain are at risk whenthey consume pesticide contaminated crops or insects. The handling andapplication of chemical pesticides also presents exposure danger to thepublic and professionals, and could lead to accidental dispersal intounintended environmentally sensitive areas. In addition, prolongedchemical pesticide application may result in an insect populationbecoming resistant to a chemical pesticide. In order to control atraditionally chemical resistant-pest, new more potent chemicalpesticides must be utilized, which in turn will lead to anotherresistance cycle. As such, there is a need in the art to control pestpopulations without the disadvantages of traditional chemicalpesticides.

An approach to decrease dependence on chemical pesticides is by causinga specific gene(s) of the target-pest to malfunction by either overexpression or silencing gene expression. The silencing approach utilizesRNA interference pathways to knockdown the gene of interest via doublestranded RNA. Double stranded RNA (dsRNA) induces sequence—specificpost-transcriptional gene silencing in many organisms by a process knownas RNA interference (RNAi). RNAi is a post-transcriptional, highlyconserved process in eukaryotes that leads to specific gene silencingthrough degradation of the target mRNA. The silencing mechanism ismediated by dsRNA that is homologous in sequence to the gene ofinterest. The dsRNA is processed into small interfering RNA (siRNA) byan endogenous enzyme called DICER inside the target pest, and the siRNAsare then incorporated into a multi-component RNA-induced silencingcomplex (RISC), which finds and cleaves the target mRNA. The dsRNAinhibits expression of at least one gene within the target, which exertsa deleterious effect upon the target.

Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process ofintroducing RNA into a living cell to inhibit gene expression of atarget gene in that cell. The RNA has a region with double-strandedstructure. Inhibition is sequence-specific in that the nucleotidesequences of the duplex region of the RNA and of a portion of the targetgene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559)discloses a method to inhibit expression of a target gene in a cell, themethod comprising introduction of a double stranded ribonucleic acidinto the cell in an amount sufficient to inhibit expression of thetarget gene, wherein the RNA is a double-stranded molecule with a firstribonucleic acid strand consisting essentially of a ribonucleotidesequence which corresponds to a nucleotide sequence of the target geneand a second ribonucleic acid strand consisting essentially of aribonucleotide sequence which is complementary to the nucleotidesequence of the target gene. Furthermore, the first and the secondribonucleotide strands are separately complementary strands thathybridize to each other to form the said double-stranded construct, andthe double-stranded construct inhibits expression of the target gene.

To utilize RNA interference as a method to regulate gene expression tocontrol a target organism, a specific essential gene needs to betargeted. One such gene is the Chloride Intracellular Channel (CLIC)gene. The CLIC gene encodes a multifunctional protein thought to beinvolved in number of cellular processes based on its role inglutathione signaling and in allowing chloride ion flux across membranes(Averaimo, et al. 2010).

Another gene of interest is Sucrase. The Sucrase gene encodes a proteinresponsible for the hydrolysis of sucrose into fructose and glucose.Sucrose is the main plant sugar, and so is the most important sustenancefor plant-feeding insects. Interference with an insect's ability tometabolize sucrose will thereby starve the insect (Karley, et al. 2005).

Such novel control methods that would induce silencing of CLIC andSucrase would be desirable as they avoid the undesirable characteristicsof traditional chemical pesticides. Traditional chemical pesticides ingeneral have the disadvantage of being toxic to the environment as wellas affecting a broad range of insect. To that end, there is a need todevelop dsRNA constructs that are engineered to target and silence CLICand Sucrase mRNA that would overcome some of the disadvantages of usingtraditional pesticides and that can target specific pests.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a double-stranded ribonucleic acid (dsRNA) forinhibiting the expression of a Chloride Intracellular Channel protein ina cell, wherein said dsRNA comprises a sense strand comprising a firstsequence and an antisense stand comprising a second sequencecomplementary to SEQ ID NO: 5, wherein said first sequence iscomplementary to said second sequence.

Also disclosed herein is a double-stranded ribonucleic acid (dsRNA) forinhibiting the expression of a Sucrase enzyme in a cell, wherein saiddsRNA comprises a sense strand comprising a first sequence and anantisense stand comprising a second sequence complementary to SEQ ID NO:10, wherein said first sequence is complementary to said secondsequence.

Disclosed herewith is a method for controlling an aphid species, themethod comprising: constructing a double stranded ribonucleic acidconstruct that is complementary to a gene that encodes a ChlorideIntracellular Channel protein, dissolving the double strandedribonucleic acid to form a solution, and contacting an effective amountof said solution to an aphid species, wherein said solution is ingestedby said aphid and RNA interference is induced, resulting in mortality ofsaid aphid.

In one embodiment of the invention, one strand of the double strandedribonucleic acid is used to control the aphid, wherein the doublestranded ribonucleic acid is complementary to the nucleotide sequence ofSEQ ID NO: 5.

Disclosed herewith is a method for controlling an aphid species, themethod comprising: constructing a double stranded ribonucleic acidconstruct that is complementary to a gene that encodes a Sucrase enzyme,dissolving the double stranded ribonucleic acid to form a solution, andcontacting an effective amount of said solution to an aphid species,wherein said solution is ingested by said aphid and RNA interference isinduced, resulting in mortality of said aphid.

In one embodiment of the invention, one strand of the double strandedribonucleic acid is used to control the aphid, wherein the doublestranded ribonucleic acid is complementary to the nucleotide sequence ofSEQ ID NO: 10.

In another embodiment of the invention, the dsRNA construct disclosedherein was used to control aphid species that consist essentially ofDiuraphis noxia, Myzus persicae, and Schizaphis graminum.

In another embodiment of the invention, the double stranded ribonucleicacid construct is dissolved in a sucrose solution. In yet anotherembodiment of the invention, the double stranded ribonucleic acidconstruct is dissolved in water.

In an embodiment of the invention, the double stranded ribonucleic acidconstruct is applied to aphid bait material. In various embodiments ofthe bait material, the bait material is a granular bait. In differentembodiments of the bait material, the bait material can be a solution orgranules that attract a target aphid.

In another embodiment of the invention, a double stranded ribonucleicacid construct is mixed with a solution, wherein the solution is appliedtopically to a plant control aphid.

It is contemplated that the dsRNA constructs disclosed herein can bedelivered to a target aphid via plant-mediated delivery. An example ofthis would be a transgenic plant expressing the dsRNA construct in theplant parts the target aphid would ingest. Another contemplated RNAiconstruct delivery means includes spraying a plant with a solution ofcomprising the dsRNA construct. Other contemplated RNAi constructdelivery means includes spraying the target aphid with a solutioncomprising the dsRNA construct wherein the solution penetrates the aphidcuticle and delivers the dsRNA construct in vivo. A contemplated exampleof this would be a surfactant solution comprising the dsRNA constructsdisclosed herein. In another embodiment, it is contemplated that thedsRNA construct can be delivered via a virus-induced gene silencing(VIGS) for transient induction of silencing. (doi:10.1101/pdb.prot5139Cold Spring Harb Protoc 2009).

BRIEF DESCRIPTION OF THE DRAWING

The present invention together with the disclosed embodiments may bestbe understood from the following detailed description of the drawings,wherein:

FIGS. 1A-C are graphs depicting mortality percentages of Diuraphisnoxia, Myzus persicae, and Schizaphis graminum over a period of 13 daysafter liquid feeding assay initiated with in vitro synthesizeddsRNA-CLIC construct compared with a control GFP dsRNA.

FIGS. 2A-C are graphs depicting reduced feeding time of Diuraphis noxia,Myzus persicae, and Schizaphis graminum over a period of 13 days afterliquid feeding assay initiated with in vitro synthesized dsRNA-CLICconstruct compared with a control GFP dsRNA.

FIGS. 3A-C are graphs depicting mortality percentages of Diuraphisnoxia, Myzus persicae, and Schizaphis graminum over a period of 13 daysafter liquid feeding assay initiated with in vitro synthesizeddsRNA-Sucrase construct compared with a control GFP dsRNA.

FIGS. 4A-C are graphs depicting reduced feeding time of Diuraphis noxia,Myzus persicae, and Schizaphis graminum over a period of 13 days afterliquid feeding assay initiated with in vitro synthesized dsRNA-Sucraseconstruct compared with a control GFP dsRNA.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is CLIC-F primer, TGGTAATGGGCACGAAGAA.

SEQ. ID NO: 2 is CLIC-R primer, AATCGGGAGGAGGTTTTTG.

SEQ. ID NO: 3 is T7_CLIC-F primer,TAATACGACTCACTATAGGGAGAATGGTAATGGGCACGAAGAA.

SEQ. ID NO: 4 is T7_CLIC-R primer,TAATACGACTCACTATAGGGAGAAAATCGGGAGGAGGTTTTTG.

SEQ. ID NO 5 is the sense strand of a 5′ to 3′ dsRNA product referred toas dsRNA-CLIC: ATGGTAATGGGCACGAAGAAAATGGACATGTGCCGGAAATTGAGCTCATCATTAAGGCTTCAACAATTGACGGTCGACGAAAAGGAGCATGTCTATTTTGCCAAGAATATTTCATGGACCTATATCTACTTGCCGAGCTAAAAACCATCAGTCTTAAAGTCACTACAGTAGATATGCAAAAACCTCCTCCCGATTT.

SEQ. ID NO: 6 is Sucrase-F primer, TCACGTACTATGCCGACGAG.

SEQ. ID NO: 7 is Sucrase-R primer, GAAGCCACGTTCCATTTGTT.

SEQ. ID NO: 8 is T7_Sucrase-F primer,TAATACGACTCACTATAGGGAGATCACGTACTATGCCGACGAG.

SEQ. ID NO: 9 is T7_Sucrase-R primer,TAATACGACTCACTATAGGGAGAGAAGCCACGTTCCATTTGTT.

SEQ. ID NO: 10 is the sense strand of a 5′ to 3′ dsRNA product referredto as dsRNA-Sucrase:TCACGTACTATGCCGACGAGTTGGGTGTACAAAACACGTATGTCCGATGGAACCAAACTGTGGACCCGGCAGGACGTAACGTCGGTCCATTGCGGTACACGCAATTCACTAGAGATCCAGCCAGAACTCCGTTTCCCTGGAATGACTCTGAAAACGCAGGTTTTACTAACGGAACAAATGGAACGTGGCTTC.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are specific aphid dsRNA constructs that target eitherChloride Intracellular Channel (CLIC) gene expression or Sucrase geneexpression. Also disclosed is the use of dsRNA constructs of a CLIC geneto interfere with critical functions of CLIC gene peptide products. Anovel method to develop nucleic acid control for pest management is alsodisclosed. Also disclosed is the use of dsRNA constructs to interferewith critical functions of Sucrase gene peptide products.

Definitions

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The term “gene” refers to a DNA sequence involved in producing apolypeptide or precursor thereof. The polypeptide can be encoded by afull-length coding sequence or by any portion of the coding sequence,such as exon sequences.

The term “oligonucleotide” refers to a molecule comprising a pluralityof deoxyribonucleotides or ribonucleotides. Oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, polymerase chain reaction, or a combinationthereof. The present invention embodies utilizing the oligonucleotide inthe form of dsRNA as means of interfering with native gene expressionthat leads to control of the target insect. Inasmuch as mononucleotidesare synthesized to construct oligonucleotides in a manner such that the5′ phosphate of one mononucleotide pentose ring is attached to the 3′oxygen of its neighbor in one direction via a phosphodiester linkage, anend of an oligonucleotide is referred to as the “5′ end” if its 5′phosphate is not linked to the 3′ oxygen of a mononucleotide pentosering and as the “3′ end” if its 3′ oxygen is not linked to a 5′phosphate of a subsequent mononucleotide pentose ring. As used herein, anucleic acid sequence, even if internal to a larger oligonucleotide,also may be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide, which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

A primer is selected to be “substantially complementary” to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence is sufficiently complementarywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

The term “double stranded RNA” or “dsRNA” refers to two substantiallycomplementary strands of ribonucleic acid. “Identity,” as used herein,is the relationship between two or more polynucleotide sequences, asdetermined by comparing the sequences. Identity also means the degree ofsequence relatedness between polynucleotide sequences, as determined bythe match between strings of such sequences. Identity can be readilycalculated (see, e.g, Computation Molecular Biology, Lesk, A. M., eds.,Oxford University Press, New York (1998), and Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York (1993),both of which are incorporated by reference herein). While there exist anumber of methods to measure identity between two polynucleotidesequences, the term is well known to skilled artisans (see, e.g.,Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J.,eds., M Stockton Press, New York (1991)). Methods commonly employed todetermine identity between sequences include, for example, thosedisclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988)48:1073. “Substantially identical” as used herein, means there is a veryhigh degree of homology (preferably 100% sequence identity) between theinhibitory dsRNA and the corresponding part of the target gene. However,dsRNA having greater than 90% or 95% sequence identity may be used inthe present invention, and thus sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence can be tolerated. Although 100% identity is preferred, thedsRNA may contain single or multiple base pair random mismatches betweenthe RNA and the target gene, provided that the mismatches occur at adistance of at least three nucleotides from the fusion site.

As used herein, “target gene” refers to a section of a DNA strand of adouble-stranded DNA that is complementary to a section of a DNA strand,including all transcribed regions, that serves as a matrix fortranscription. The target gene is therefore usually the sense strand.

The term “complementary RNA strand” refers to the strand of the dsRNA,which is complementary to an mRNA transcript that is formed duringexpression of the target gene, or its processing products. “dsRNA”refers to a ribonucleic acid molecule having a duplex structurecomprising two complementary and anti-parallel nucleic acid strands. Notall nucleotides of a dsRNA must exhibit Watson-Crick base pairs. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA.

“Small interfering RNA” or “siRNA” refers to a short double-strand ofribonucleic acid, approximately 18 to 200 nucleotides in length. Theterm “RNA interference” or “RNAi” refers to a cellular mechanism for thedestruction of targeted ribonucleic acid molecules. Under endogenousconditions, RNAi mechanism operates when dsRNA is cleaved to siRNA viaan enzyme, DICER. The siRNA is processed to a single strand ofanti-sense ribonucleic acid and coupled with a protein complex namedRISC. The antisense RNA then targets a complementary gene construct,such as messenger RNA that is cleaved by ribonuclease. While theexamples infra discloses constructing dsRNA constructs via enzymatictechniques with the enzyme RNA polymerase, it is contemplated that siRNAcan be constructed via RNA oligonucleotide synthesis such as thosedisclosed in Scaringe, S., Methods Enzymol., 2000, Vol. 317:3 andincorporated herein by reference.

Disclosed herein are long dsRNA constructs, such as the SEQ ID NOS: 5and 10. It is contemplated that siRNA and /or partial dsRNA sequencesfrom those sequence listings constructs comprising variousdouble-stranded base pairs of disclosed long dsDNA constructs would beeffective in knocking-down the gene function in a target aphid species.It is contemplated that such siRNA and/or partial dsRNA sequences fromSEQ ID NOS: 5 and 10 could be generated synthetically or enzymaticallyin accordance with the teachings herein.

As used herein, “knock-down” is defined as the act of binding anoligonucleotide with a complementary nucleotide sequence of a gene assuch that the expression of the gene or mRNA transcript decreases.

The term “substantially single-stranded” when used in reference to anucleic acid product means that the product molecule exists primarily asa single strand of nucleic acid in contrast to a double-stranded productwhich exists as two strands of nucleic acids which are held together byinter-strand base pairing interactions.

“Oligonucleotide primers matching or complementary to a gene sequence”refers to oligonucleotide primers capable of facilitating thetemplate-dependent synthesis of single or double-stranded nucleic acids.Oligonucleotide primers matching or complementary to a gene sequence maybe used in PCRs, RT-PCRs and the like.

The term “corresponds to” as used herein means a polynucleotide sequencehomologous to all or a portion of a reference polynucleotide sequence,or a polypeptide sequence that is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For example, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

An “effective amount” is an amount sufficient to effect desiredbeneficial or deleterious results. An effective amount can beadministered in one or more administrations. In terms of treatment, an“effective amount” is that amount sufficient to make the target pestnon-functional by causing an adverse effect on that pest, including (butnot limited to) physiological damage to the pest; inhibition ormodulation of pest growth; inhibition or modulation of pestreproduction; or death of the pest. In one embodiment of the invention,a dsRNA containing solution is fed to a target insect in an amount ofapproximately at a concentration of 314 ng/μl of solution. An effectiveamount include amounts less that that concentration in which pestmortality would still occur.

The term “solvent” includes any liquid that holds another substance insolution. Examples of solvents include but are not limited to water andorganic solvents such as acetone, ethanol, dimethyl sulfoxide (DMSO),and dimethylformamide (DMF).

As used herein, the term “GFP dsRNA” refers to a control dsRNAconstruct. The green fluorescent protein (GFP) is commonly used as areporter gene and was originally isolated from jellyfish and widely usedas control in prokaryotic and eukaryotic systems.

The term “phagostimulant” refers to any substance that will entice theinsect to ingest the dsRNA. For insects, suitable phagostimulantsinclude but are not limited to edible oils and fats, vegetable seedmeals, meal by-products such as blood, fish meal, syrups, honey, aqueoussolutions of sucrose, artificial sweeteners such as sucralose,saccharin, and other artificial sweeteners, peanut butter, cereals,amino acids, and other proteins. Sucrose is a will known feedingstimulant for aphids.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding Guaninenucleotide binding protein gene and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y, and Ausubel, F. M. et al. (2001) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.

While the examples provided herein describe dsRNA constructs cloned fromGenBank Accession No. JOTR00000000 it is contemplated that when read inconjunction with the teaching disclosed herein, the construction ofother dsRNA constructs targeting CLIC or Sucrase gene sequences of otherinsect orders would be feasible to those skilled the in the art.Additionally it is contemplated that a single dsRNA construct would beeffective in controlling a plurality of insect species.

Aphid colonies used in the foregoing examples originated from fieldcollections held at USDA, ARS, Stillwater, Okla. The biotype E greenbugand Russian wheat aphid biotype 2 (RWA2) were both reared on ‘Yuma’wheat 6 months prior to tests. Myzus persicae was reared on a mixture of‘Long Island Imperial’ brussel sprouts and ‘Southern Giant’ mustard.Aphid host plants were grown potting soil in 8 cm diameter pots coveredby ventilated cylindrical clear plastic cages. Aphid infested plantswere held in a room with temperatures of 20-22° C. on light racks with a14:10 L:D photoperiod provided by four 40 w cool white fluorescentlights. As referred to herein, “RWA” refers to the Russian wheat aphid.It is contemplated that the dsRNA constructs disclosed herein would havebroad aphid activity against aphids, a large range of taxonomicallydistant aphid species including but not limited to: the greenbug,Schizaphis graminum; green peach aphid, Myzus persicae; Russian wheataphid, Diuraphis noxia; pea aphid, Acyrthosiphon pisum; soybean aphid,Aphis glycines; and bird cherry-oat aphid, Rhopalosiphum padi.

EXAMPLE 1 Constructing dsRNA Construct for Aphid Species

Cloning and Sequencing of the CLIC Gene

The CLIC gene was identified by comparing RNA expressed by RWA, asdetermined by RNAseq expression data, to the RWA whole genome sequence.The CLIC mRNA sequence was identified and primers were created for itsamplification and for production of sequence-specific dsRNA. To createdsRNA specifically targeting the RWA CLIC sequence, whole RNA wasisolated from 50 adult RWA biotype 2 aphids using the Promega SV totalRNA isolation kit. Aphids were ground together in liquid nitrogen andRNA extraction proceeded according to protocol. Total RNA was eluted inRNAse-free water (Promega), DNAse-treated (Ambion DNA-free kit), andquantified and assessed for purity using a spectrophotometer (A260). RNAaliquots were stored at −80° C. cDNA of CLIC was amplified from totalRNA using first-strand synthesis with gene-specific reverse primer(CLIC-R (Seq 2)) (Agilent Affinity-script Multi-Temperature cDNAsynthesis kit). The first-strand synthesis reaction was then used as atemplate for end-point PCR using the full (CLIC-F(Seq 1) and CLIC-R(Seq2)) primer set. The full-length PCR product for CLIC was thengel-purified (Promega SV Gel and PCR Clean-up System) and subjected toPCR using primers with attached T7 RNA polymerase promoters(T7_CLIC-F(Seq 3) and T7_CLIC-R (Seq 4)). This fragment, which possessedT7 RNA polymerase promoters on each 5′ end (sense and antisense strand),was gel-purified and stored at −80° C.

Construction of CLIC dsRNA Constructs

The T7-containing CLIC fragments generated as described in the passageabove were used as a template to generate double-stranded RNA afterspectrophotometric quantification (A260). 200 ng of the CLIC T7 templatewas used in each reaction to generate dsRNA copies of CLIC with theAmbion Megascript in vitro transcription kit. The reactions were allowedto proceed for 16 hours at 37° C. and the resulting product was columnpurified with Ambion Nuc-Away columns, quantified spectrophotometrically(A260) and visualized on an agarose gel to assess purity andconcentration. The isolated dsRNA was then stored at −80° C. forpreservation until further use.

EXAMPLE 2 dsRNA Construct Feeding Bioassay Using Liquid Bait Station

All aphid species were fed upon the same artificial diet consisting of15% sucrose solution in water amended with a 2% amino acid solution(‘Complete Amino Acid Mix’, Nutricia North America, Rockville, Md.) andaddition of 314 ng per μl of each dsRNA. The dsRNA treatments were mixedwithin the artificial diet at a concentration of 314 ng per μl for CLIC.The dsRNA treated and null control diets were contained in feedingstations consisting of a 35 mm diam. petri dish 10 mm deep×35 with 50 ulof diet sandwiched between two layers of stretched parafilm. A 15 mmrubber o-ring was placed over the diet area, 20-30 aphids were placed onthe diet within the ring, and sealed within the o-ring by the petri dishlid held in place with parafilm. This feeding station allowed the aphidsto move and feed freely for up to 15 d. The clear plastic lid thatsealed the aphids on the diet allowed aphid and mobility counts to bemade without lid removal. The toxicity of CLIC dsRNAs was determined bypercent mortality, and the number of aphids actively feeding wascompared between dsRNA treatments and null controls every two days up to13 d post infestation. Unsettled/agitated aphids that were not activelyfeeding usually died within 7 d after being placed on successful dsRNAconstructs that showed activity against aphids versus other dsRNAconstructs that had no activity.

EXAMPLE 3 Constructing dsRNA Construct for Aphid Species

Cloning and Sequencing of Sucrase Gene

The Sucrase gene was identified by comparing expressed RNA in RWA, asdetermined by RNAseq expression data, to the RWA whole genome sequence.The Sucrase mRNA sequence was identified and primers were created forits amplification and for production of sequence-specific dsRNA. Tocreate dsRNA specifically targeting the RWA Sucrase sequence, whole RNAwas isolated from 50 adult RWA biotype 2 aphids using the Promega SVtotal RNA isolation kit. Aphids were ground together in liquid nitrogenand RNA extraction proceeded according to protocol. Total RNA was elutedin RNAse-free water (Promega), DNAse-treated (Ambion DNA-free kit), andquantified and assessed for purity using a spectrophotometer (A260). RNAaliquots were stored at −80° C. cDNA of Sucrase was amplified from totalRNA using first-strand synthesis with gene-specific reverse primer(Sucrase-R (Seq7)) (Agilent Affinity-script Milti-Temperature cDNAsynthesis kit). The first-strand synthesis reaction was then used as atemplate for end-point PCR using the full (Sucrase-F (SEQ 6) andSucrase-R(SEQ7)) primer set. The full-length PCR product for Sucrase wasthen gel-purified (Promega SV Gel and PCR Clean-up System) and subjectedto PCR using primers with attached T7 RNA polymerase promoters(T7_Sucrase-F (SEQ8) and T7_Sucrase-R (SEQ 9)). This fragment, whichpossessed T7 RNA polymerase promoters on each 5′ end (sense andantisense strand) was gel-purified and stored at −80° C.

Construction of Sucrase dsRNA Constructs

The T7-containing Sucrase fragments generated as described in thepassage above were used as a template to generate double-stranded RNAafter spectrophotometric quantification (A260). 200 ng of the Sucrase T7template was used in each reaction to generate dsRNA copies of Sucrasewith the Ambion Megascript in vitro transcription kit. The reactionswere allowed to proceed for 16 hours at 37° C. and the resulting productwas column purified with Ambion Nuc-Away columns, quantifiedspectrophotometrically (A260) and visualized on an agarose gel to assesspurity and concentration. The isolated dsRNA was then stored at −80° C.for preservation until further use.

EXAMPLE 4 dsRNA Construct Feeding Bioassay Using Liquid Bait Station

All aphid species were fed upon the same artificial diet consisting of15% sucrose solution in water amended with an amino acid solutioncontaining 314 ng per μl of Sucrase dsRNA. The dsRNA treatments weremixed within the artificial diet at a concentration of 314 ng per μl forSucrase. The dsRNA treated and null control diets were contained infeeding stations consisting of a 35 mm diam. petri dish 10 mm deep×35with 50 ul of diet sandwiched between two layers of stretched parafilm.A 15 mm rubber o-ring was placed over the diet area, 20-30 aphids wereplaced on the diet within the ring, and sealed within the o-ring by thepetri dish lid held in place with parafilm. This feeding station allowedthe aphids to move and feed freely for up to 15 d. The clear plastic lidthat sealed the aphids on the diet allowed aphid and mobility counts tobe made without lid removal. The toxicity of Sucrase dsRNA wasdetermined by percent mortality, and the numbers of aphids activelyfeeding were compared between Sucrase dsRNA treatments and null controlsevery two days up to 13 d post infestation. Unsettled/agitated aphidsthat were not actively feeding usually died within 7 d after beingplaced on successful dsRNA constructs that showed activity againstaphids versus other dsRNA constructs that had no activity.

To the extent that the term “includes” or “including” is employed in thedetailed description or the claims, it is intended to be inclusive in amanner similar to the term “comprising” as that term is interpreted whenemployed as a transitional word in a claim. Furthermore, to the extentthat the term “or” is employed in the detailed description or claims(e.g., A or B) it is intended to mean “A or B or both”. When theapplicants intend to indicate “only A or B but not both” then the term“only A or B but not both” will be employed. Thus, use of the term “or”herein is the inclusive, and not the exclusive use. See, Bryan A.Garner, A Dictionary of Modern Legal Usage 624 (2d. Ed. 1995). Also, tothe extent that the terms “in” or “into” are used in the specificationor the claims, it is intended to additionally mean “on” or “onto.”Furthermore, to the extent the term “connect” is used in thespecification or claims, it is intended to mean not only “directlyconnected to,” but also “indirectly connected to” such as connectedthrough another component or components.

While the invention has been described with reference to details of theillustrated embodiment, these details are not intended to limit thescope of the invention as defined in the appended claims. All citedreferences and published patent applications cited in this applicationare incorporated herein by reference. The embodiment of the invention inwhich exclusive property or privilege is claimed is defined as follows:

The invention claimed is:
 1. A double-stranded ribonucleic acid (dsRNA)for inhibiting the expression of a chloride intracellular channelprotein (CLIC) in a cell, wherein said dsRNA comprises a first strandcomprising a sequence with at least 95% sequence identity to a portionof at least 18 consecutive nucleotides of SEQ ID NO: 5 and a secondstrand complementary to the first strand.
 2. A method for controlling anaphid, the method comprising: constructing a double stranded ribonucleicacid (dsRNA) wherein one strand of the dsRNA comprises a sequence withat least 95% sequence identity to a portion of at least 18 consecutivenucleotides of SEQ ID NO: 5 that is complementary to an aphid gene thatencodes a chloride intracellular channel protein, dissolving the dsRNAto form a solution, and contacting an effective amount of said solutionto an aphid species, wherein said solution is ingested by said aphidspecies and RNA interference is induced, resulting in mortality of saidaphid.
 3. The method of claim 2, wherein the double stranded ribonucleicacid construct is dissolved in a sucrose solution.
 4. The method ofclaim 2, wherein the double stranded ribonucleic acid construct isdissolved in water.
 5. The method of claim 2, wherein the solution isapplied to aphid bait material.
 6. The method of claim 2, wherein thesolution further comprises a surfactant.
 7. The method of claim 2,wherein the solution further comprises a virus-induced gene silencing(VIGS) vector.
 8. An aphid control solution comprising a dsRNA, thedsRNA comprising a first sequence with at least 95% sequence identity toa portion of at least 18 consecutive nucleotides of SEQ ID NO: 5 and asecond strand complementary to the first strand, wherein the dsRNA isdissolved in a solution and the solution optionally comprises asurfactant, a virus-induced gene silencing (VIGS) vector, or both asurfactant and a VIGS vector.
 9. The method of claim 2, wherein theeffective amount of double stranded ribonucleic acid is approximately314 ng per μl.
 10. The method of claim 2, wherein the aphid species isDiuraphis noxia, Myzus persicae, or Schizaphis graminum.